Therapeutic use of compounds

ABSTRACT

A compound having the formula (I) R1-COOH wherein R1 is an alkyl or alkenyl group having a C7-11 backbone, optionally branched with a C1-6 alkyl group at any C position in the backbone, or a pharmaceutically acceptable salt, amide or ester thereof, wherein the backbone of the alkyl or alkenyl group, and/or the branched alkyl groups, are optionally interrupted by one or more heteroatoms, provided that when R1 is an alkyl group having a C7 backbone, the branching does not consist only of a hexyl group at the a carbon of R1, or only of a methyl group at the γ carbon of R1, or of only single methyl groups at both the β and ω−1 carbons of R1, and provided that when R1 is an alkyl group having a C8 or C11 backbone, the branching does not consist only of a propyl group at the a carbon of R1, for use in the treatment or prevention of a disease or a biomedical condition selected from seizure-related disorders, bipolar disorders, mania, depression, migraine, attention deficit hyperactivity disorders, latent HIV infection, Alzheimer&#39;s disease, chorea, schizophrenia, ischemia, cancer and fatal blood loss.

This invention relates to uses of compounds. In particular, it relatesto the use of compounds in the treatment or prevention of diseases andbiomedical conditions such as, seizure-related disorders, bipolardisorders, mania, depression, migraine, attention deficit hyperactivitydisorders, latent HIV infection, Alzheimer's disease, chorea andschizophrenia, ischemia, cancer and fatal blood loss.

Epilepsy is a widespread, serious neurological condition presentingconsiderable personal, social and economic difficulty. It affects 0.5-1%of the population, of which 30% have epilepsy that is not adequatelytreated with present antiepileptic drugs (Bialer and White 2010). Thesepatients have a high mortality and morbidity rate. The understanding ofcellular and molecular aspects of seizures giving rise to epilepsy isunclear, although current research has centered on defining themolecular pathways necessary for seizure progression and the developmentof new treatments for seizure control.

Valproic acid (VPA; 2-propylpentanoic acid; Epilim®), a short chainedbranched fatty acid, is the most widely used anti-epileptic world-wide,but its mechanism of action in seizure control has remained relativelyunclear for over 40 years (Lagace et al., 2005; Perucca, 2002). Havingbeen accidentally found to be effective in seizure control (Carraz G.,1967), VPA is now also used for bipolar disorders and migrainetreatment, in addition to a variety of potential new therapies includingcancer and HIV treatment. VPA has previously been shown to have achronic effect in controlling inositol depletion (Williams et al., 2006;Williams, 2005), and this long-term effect is likely to be related toits efficacy in bipolar disorders.

With regard to epilepsy treatment, the therapeutic effects of VPA havebeen proposed to occur via directly elevating gamma-amino butyric acid(GABA) signalling (Lagace et al., 2005) and inhibiting sodium channelactivity (Costa et al., 2006). Of prime importance in VPA's mechanism ofaction in epilepsy treatment is that it blocks seizure activityacutely—within 30 minutes of administration—corresponding to the peakconcentration of VPA in the brain following intravenous injection (Alyand bdel-Latif, 1980). Despite such rapid action suggesting a directaction on channels or a biochemically-based (rather thantranscriptionally-based) epilepsy target, few acute effects of VPA havebeen identified (Lagace et al., 2005), making rapid VPA-catalysedeffects of great potential therapeutic importance. The acute effect ofVPA was recently analysed using the simple model Dictyostelium (Xu etal., 2007). It demonstrated that VPA induced an inhibition ofphosphatidylinositol-(3,4,5)-trisphosphate (PIP₃) production and areduction in phosphatidylinositol monophosphate (PIP) and diphosphate(PIP₂) phosphorylation.

Bipolar disorder post mortem brain samples also show altered levels ofenzymes associated with fatty acid turnover (Kim et al., 2009) as wellas altered fatty acids within cell membranes (Chiu et al., 2003).Numerous studies have also shown an increase in arachidonic acid (AA)release after seizure catalysed by increased PLA₂ activity (Siesjo etal., 1982, Rintala et al., 1999, Bazan et al., 2002, Basselin et al.,2003), and attenuation of this process may thus provide some benefit inseizure control and epileptogenesis (Rapoport and Bosetti, 2002).

VPA has been shown to reduce AA turnover in the brain through an unknownmechanism (Chang et al., 2001). AA is an essential fatty acid and is themajor polyunsaturated fatty acid in most membrane phospholipids(Svennerholm, 1968), and plays a central role in inflammatory signalling(Yedgar et al., 2006). It remains unclear if the effect of VPA on AAturnover is related to specific VPA-treatable conditions. For example,this effect may be related to bipolar disorder prophylaxis (Rapoport,2008b) since a similar reduction in AA signalling has also been observedwith other structurally independent bipolar disorder treatments such aslithium (Basselin et al., 2005) and carbamazepine (Bazinet et al.,2006a).

Although widely prescribed for multiple diseases, VPA has a number ofunwanted side effects including teratogenicity and hepatotoxicity.Therefore, more potent antiepileptic drugs with reduced side effects areurgently needed.

International patent application WO 99/02485 discloses a family of VPAanalogs for treating epilepsy, migraine, bipolar disorders and pain. Thecompounds specifically disclosed in WO 99/02485 are 2-propylheptylaceticacid, 2-propyldecanyl acetic acid and1-O-stearoyl-2-propylheptylacetoyl-sn-glycero-3-phosphotidylcholine.

Using the biomedical model Dictyostelium, the inventors found that theeffect of VPA is to cause a rapid attenuation of phosphoinositideturnover, and this effect is not based upon the direct inhibition ofphosphatidylinositol-3-kinase (PI3K) activity, nor is it caused throughregulation of inositol recycling. They also found that VPA induced botha reduced release and an increased uptake of radiolabelled AA andpalmitic acid (a saturated long chain fatty acid). This VPA-catalysedeffect is not caused by reducing fatty acid activation.

In addition, structure-activity relationship (SAR) studies showed a highdegree of structural specificity for these mechanisms of action. Thisenabled the identification of a group of compounds showing therapeuticpotential similar to VPA but with the potential for reduced side effectsand/or increased therapeutic efficacy.

In accordance with a first aspect of the present invention, there isprovided a compound having the FormulaR1-COOH  (I)wherein R1 is an alkyl or alkenyl group having a C₇₋₁₁ backbone,optionally branched with a C₁₋₆ alkyl group at any C position in thebackbone, or a pharmaceutically acceptable salt, amide or ester thereof,wherein the backbone of the alkyl or alkenyl group, and/or the branchedalkyl groups, are optionally interrupted by one or more heteroatoms,provided that when R1 is an alkyl group having a C₇ backbone, thebranching does not consist only of a hexyl group at the α carbon of R1,or only of a methyl group at the γ carbon of R1, or of only singlemethyl groups at both the β and ω-1 carbons of R1, and provided thatwhen R1 is an alkyl group having a C₈ or C₁₁ backbone, the branchingdoes not consist only of a propyl group at the α carbon of R1,for use in the treatment or prevention of a disease or a biomedicalcondition selected from a seizure-related disorders, bipolar disorders,mania, depression, migraine, attention deficit hyperactivity disorders,latent HIV infection, Alzheimer's disease, chorea, schizophrenia,ischemia, cancer and fatal blood loss, provided that, when the compoundis 2-methyl-2-pentenoic acid, the disease or condition is not bipolardisorder or epilepsy.

The compounds described herein have been found to cause rapidattenuation of phosphoinositol turnover and/or attenuation of fatty acidturnover. Since attenuation of phosphoinositol and fatty acid turnoverhave been identified as mechanisms of action of VPA, these compounds mayhave the potential to be useful in the treatment or prevention ofVPA-treatable conditions, such as seizure-related disorders, bipolardisorders, mania, depression, migraine, attention deficit hyperactivitydisorders, latent HIV infection, Alzheimer's disease, chorea andschizophrenia, in particular, epilepsy, bipolar disorders and migraine.

In an embodiment, when R1 is an alkyl group having a C₇ backbone, thebranching does not consist only of a methyl group at the ω-1 carbon ofR1, and preferably does not comprise a methyl group at the ω-1 carbon ofR1.

Compounds that can be used for the purpose of the invention include, butare not limited to, nonanoic acid, decanoic acid, 4-ethyloctanoic acid,2-propyloctanoic acid, 2-butyloctanoic acid, 4-methylnonanoic acid,8-methylnonanoic acid, 3-methylnonanoic acid, and 3-Methylundecanoicacid.

The term ‘an alkyl group having a C_(x-y) backbone’ as used hereinrefers to a linear saturated hydrocarbon group containing from x to ycarbon atoms. For example, an alkyl group having a C₁₋₄ backbone refersto an unbranched saturated hydrocarbon group containing from 1 to 4carbon atoms. Examples of an alkyl group having a C₁₋₄ backbone includemethyl, ethyl, propyl, and butyl.

The term ‘an alkenyl group having a C_(x-y) backbone’ as used hereinrefers to a linear unsaturated hydrocarbon group containing from x to ycarbon atoms and at least one (e.g. 1, 2, 3 or 4) double bonds. Forexample, an alkenyl group having a C₃₋₅ backbone refers to an unbranchedunsaturated hydrocarbon group containing from 3 to 5 carbon atoms.Examples of an alkenyl group having a C₃₋₅ backbone include propylene,butylene and pentylene.

The terms “α carbon of R1”, “β carbon of R1” and “γ carbon of R1” referto the first, second and third carbon atoms, respectively, in a chain ofcarbon atoms forming R1, counting from, but not including, the COOHgroup of Formula (I). The term “ω-1 carbon of R1” refers to thepenultimate carbon atom of a chain of carbon atoms forming R1, againcounting from the COOH group of Formula (I). In other words, “ω-1 carbonof R1” is the carbon next to the terminal methyl or methylene group inR1.

The term ‘C_(x-y) alkyl’ as used herein refers to a branched orunbranched saturated hydrocarbon group containing from x to y carbonatoms. For example, C₁₋₄ alkyl refers to a branched or unbranchedsaturated hydrocarbon group containing from 1 to 4 carbon atoms.Examples of C₁₋₄ alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl and tert butyl.

‘Pharmaceutically acceptable salts’ of compounds of the presentinvention include salts with inorganic bases, salts with organic bases,salts with inorganic acids, salts with organic acids and salts withbasic or acidic amino acids. Salts with bases may, in particular, beemployed in some instances. The compound of the present invention may bein either hydrate or non-hydrate form.

‘Pharmaceutically acceptable amides’ of compounds of the presentinvention are derivatives in which the carboxyl (i.e. —C(O)OH) groups ofthe said compounds are modified by reaction with an amine —NHR1′R2′ soas to yield —C(O)NR1′R2′ groups, wherein R1′ and R2′ are optionallyindependently selected from H, C₁₋₈ alkyl (eg C₁₋₆ alkyl), aryl,heteroaryl and C₃₋₈ cycloalkyl group.

‘Pharmaceutically acceptable esters’ of compounds of the presentinvention are derivatives in which the carboxyl (i.e. —C(O)OH) groups ofthe said compounds are modified by reaction with an alcoholic moietyW—OH so as to yield —C(O)OW groups, wherein W may be C₁₋₁₈ alkyl (e.g.C₁₋₆ alkyl), aryl, heteroaryl, or C₃₋₈ cycloalkyl.

General methods for the preparation of salts, amides and esters are wellknown to the person skilled in the art. Pharmaceutical acceptability ofsalts, amides and esters will depend on a variety of factors, includingformulation processing characteristics and in vivo behaviour, and theskilled person would readily be able to assess such factors havingregard to the present disclosure.

Where compounds of the invention exist in different enantiomeric and/ordiastereoisomeric forms (including geometric isomerism about a doublebond), these compounds may be prepared as isomeric mixtures orracemates, although the invention relates to all such enantiomers orisomers, whether present in an optically pure form or as mixtures withother isomers. Individual enantiomers or isomers may be obtained bymethods known in the art, such as optical resolution of products orintermediates (for example chiral chromatographic separation (e.g.chiral HPLC)), or an enantiomeric synthesis approach. Similarly, wherecompounds of the invention may exist as alternative tautomeric forms,the invention relates to the individual tautomers in isolation, and tomixtures of the tautomers in all proportions.

In certain embodiments of the invention, R1 is an alkyl group.

In certain embodiments of the invention, R1 is an alkyl group having aC₇₋₁₀ backbone.

In certain embodiments of the invention, R1 is an alkyl group having atleast one point of branching, for example one, two or three points ofbranching.

In some embodiments, R1 is a C₇₋₁₀ backbone alkylene group comprisingbranching at any position in the backbone, preferably at the α, β, γ orω-1 carbon of R1.

In certain embodiments of the invention, the branching consists of aC₁₋₄ alkyl group, such as a methyl, ethyl, propyl or butyl group,preferably a methyl, ethyl or propyl group.

In particular embodiments, R1 is a C₇ backbone alkyl group having abranched ethyl, propyl or butyl group.

In particular embodiments, R1 is a C₈ backbone alkyl group having abranched methyl group.

In certain embodiments of the invention, R1 is an unbranched alkylgroup.

In particular embodiments, R1 is a C₈₋₉ unbranched alkyl group.

In some embodiments, the one or more heteroatoms in the alkyl or alkenylgroups is selected from the group consisting of oxygen, sulphur andnitrogen. Preferably, the one or more heteroatoms is oxygen.

In certain embodiments, the compound used for the present invention isgiven separately, simultaneously or sequentially in combination withanother pharmaceutically active agent which is known to be useful forthe treatment or prevention of a disease or a biomedical conditionselected from seizure-related disorders, bipolar disorders, mania,depression, migraine, attention deficit hyperactivity disorders, latentHIV infection, Alzheimer's disease, chorea, schizophrenia, ischemia,cancer and fatal blood loss, or co-morbidities thereof.

In certain embodiments, two or more of the compounds used in accordancewith the first aspect of the invention can be used separately,simultaneously or sequentially in combination.

In a second aspect, the invention also provides a method of treatment orprevention of a disease or a biomedical condition selected fromseizure-related disorders, bipolar disorders, mania, depression,migraine, attention deficit hyperactivity disorders, latent HIVinfection, Alzheimer's disease, chorea, schizophrenia, ischemia, cancerand fatal blood loss, in particular, epilepsy, bipolar disorders andmigraine, the method comprising the administration, to a subject in needof such treatment or prevention, of a therapeutically effective amountof a compound used according to the first aspect of the invention.

The compound may be administered with one or more conventional non-toxicpharmaceutically acceptable carrier, adjuvant or vehicle.Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in accordance with this invention are those conventionally employedin the field of pharmaceutical formulation, and include, but are notlimited to, sugars, sugar alcohols, starches, ion exchangers, alumina,aluminium stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycerine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulphate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. The compound can be administered orally, parenterally, byinhalation spray, rectally, nasally, buccally, vaginally or via animplanted reservoir. The term parenteral as used herein includessubcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intrasynovial, intrasternal, intrathecal, intralesionaland intracranial injection or infusion techniques.

The compound used in the present invention may be in administered in theform of a sterile injectable preparation, for example, as a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents (such as, for example, Tween 80) andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that maybe employed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersantsuch as that described in Ph. Helv, or a similar alcohol.

The compound used for this invention may be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, powders, granules, and aqueous suspensions and solutions. Thesedosage forms are prepared according to techniques well-known in the artof pharmaceutical formulation. In the case of tablets for oral use,carriers which are commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude lactose and dried corn starch. When aqueous suspensions areadministered orally, the active ingredient is combined with emulsifyingand suspending agents. If desired, certain sweetening and/or flavouringand/or colouring agents may be added.

The compound used for this invention may also be administered in theform of suppositories for rectal administration. For this purpose, thecompound may be mixed with a suitable non-irritating excipient which issolid at room temperature but liquid at the rectal temperature andtherefore will melt in the rectum to release the active components. Suchmaterials include, but are not limited to, cocoa butter, beeswax andpolyethylene glycols.

The compound used for this invention may be administered by nasalaerosol or inhalation. For this purpose, the compound is preparedaccording to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilising or dispersingagents known in the art.

The compounds used for the present invention may be administered in adose of around 1 to around 20,000 μg/kg per dose, depending on thecondition to be treated or prevented, and the characteristics of thesubject being administered with the compound. In many instances, thedose may be around 1 to around 1500 μg/kg per dose. The dosing regimenfor a given compound could readily be determined by the skilled personhaving access to this disclosure.

In accordance with a third aspect, the present invention provides theuse of a compound as defined according to the first aspect of theinvention in the preparation of a medicament for the treatment orprevention of a disease or a biomedical condition selected fromseizure-related disorders, bipolar disorders, mania, depression,migraine, attention deficit hyperactivity disorders, latent HIVinfection, Alzheimer's disease, chorea, schizophrenia, ischemia, cancerand fatal blood loss.

The invention will now be described in more detail by way of exampleonly with reference to the figures listed below.

FIG. 1 shows time- and dose-dependent effect of VPA in attenuation ofphosphoinositide signalling in Dictyostelium. Phosphoinositide labelingwas monitored by incorporation of a radio-labelled phosphate intonewly-formed lipids, followed by extraction, TLC separation andquantification using a Typhoon phosphorimager (Pawolleck & Williams2009). (a) Analysis of PIP and PIP₂ turnover in cells treated with VPA(0.5 mM) for indicated times. (b) Analysis of PIP and PIP₂ turnover incells treated for 9 min with varying concentrations of VPA. Results areprovided for triplicate experiments with duplicate samples±SD where*P<0.05; **P<0.01; ***P<0.001 for PIP levels.

FIG. 2 shows phosphoinositide signalling and VPA sensitivity in wildtype (wt) and knockout mutants lacking phospholipid kinase and inositolrecycling enzymes with or without 9 min VPA treatment (0.5 mM). (a)Comparison of phosphoinositide levels in untreated isogenic mutant lineslacking indicated lipid kinase activities: five type 1phosphatidylinositol 3-kinase (5×PI3K; DBS0252654) genes; thephosphatidylinositol-4-phosphate 5-kinase (rPKA; DDB0191443) gene; andthe phosphatidylinositol-4-phosphate 5-kinase gene (PIPKinA; DDB0185056)gene. VPA and PI3K inhibitor sensitivity was monitored using 0.5 mM VPAor 50 μM LY2946004 respectively for: (b) the 5×PI3K mutant; (c) the rPKAmutant and (d) the PIPKinA mutant. (e) Schematic of phosphoinositidesignalling showing the role of phospholipase C (PLC), prolyloligopeptidase (PO), inositol monophosphatase (IMPase) and myo-inositolsynthase (INO1) in the generation and recycling of phosphoinositides.(f) Ablation of PLC and PO genes did not alter VPA-attenuated PIP andPIP₂ signalling (g) Extended VPA treatment (60 min, 0.5 mM) furtherreduced phosphoinositide signalling, and this effect was not reversedfollowing overexpression of IMPase. Results are provided for triplicateexperiments with duplicate samples±SD where *P<0.05; **P<0.01;***P<0.001 for PIP levels.

FIG. 3 shows seizure control with VPA and phosphoinositide-attenuatingcompounds using in vitro acute seizure mode-pentylentetrazol (PTZ) modeland low magnesium model. (a) A combined entorhinal cortex-hippocampalslice preparation was placed in a submerged recording chamber andperfused with artificial cerebrospinal fluid containing high [K⁺] andPTZ to induce epileptiform activity prior to addition of VPA or novelcompounds at 1 mM. (b) Illustration of trace samples of burst dischargesfollowing application of VPA, 4-methyloctanoic acid (4-MO),2-propyloctanoic (2-PO), and 2-butyloctanoic acid (2-BO)). (c) Summaryof the frequency of burst discharges following application of drugsplotted against time. The drugs were applied from time 0 to 40 minutes.(d) Trace samples and (e) frequency of burst discharges for longerstraight chain nonanoic acid (NA) and decanoic acid (DA). (f)Illustration of trace samples of low magnesium-induced burst dischargesby application of VPA (1 mM), 4-methyloctanoic acid (4-MO, 1 mM). (g)Summary of the frequency of low magnesium-induced burst dischargesfollowing application of drugs (VPA 1 mM, n=5; 4-MO 1 mM, n=5). Thefrequency of epileptiform activity induced by low magnesium plottedagainst time. The drugs were applied from time 0 to 40 minutes.Application of VPA resulted in a significant decrease in dischargefrequency (72.7±3.6% of baseline, 30-40 minutes after application andthe effect of suppression is reversible after wash out), whereasapplication of 4-MO abolished the epileptiform discharge (1.6±3.1% ofbaseline, p<0.01 compared to control; P<0.01 compared to VPA). Theepileptiform activities in both treatment recovered during drug washout(n=5 for each drug). (h) Comparison of the mean frequency of lowMg2+-induced burst discharges for the last 10 min during drugapplication with different treatments, demonstrating a significanteffect of all compounds in attenuating seizure activity. * P<0.05, **P<0.01 compared to control; +P<0.05, ++P<0.01, compared to VPA treatedgroup. Data are presented as means±SEM.

FIG. 4 shows seizure control with phosphoinositide-attenuating compoundsin an in vivo seizure model. (a) Summary of the procedure—electricalinduction of self-sustained status epilepticus (SSSE). Rats wereelectrically stimulated via the perforant pathway with 4-5 mA, 50 μsmonopolar pulses at 20 Hz for 2 hours to induce SSSE seven days afterelectrode implantation. Three hours after the induction of the SSSE, therats were given diazepam (10 mg/kg) by intraperitoneal injection (i.p.)to terminate the seizure activity. (b) Illustrative trace samples of EEGfrom status epilepticus animal. Administration of VPA (400 mg/kg) or4-methyloctanoic acid (400 mg/kg) resulted in attenuation of seizureactivity, whereas DMSO had no effect. (c) Time course of the effects onspike amplitude following administration of DMSO (n=5), VPA (n=7) and4-MO (n=7). (d) Time course of the effects on spontaneous spikefrequency following administration of DMSO (n=5), VPA (n=7) or 4-MO(n=7).

FIG. 5 demonstrates that VPA induces changes in fatty acid uptake andrelease in a time and concentration dependent manner. Dictyostelium wildtype (Ax2) cells were pre-incubated with ³H arachidonic acid (A) orpalmitic acid (C) and the release of ³H into external buffer is shown inthe presence/absence of VPA. Fatty acid uptake was measured byincubation of cells with or without VPA and ³H AA (B) or palmitic acid(D) simultaneously. Uptake of ³H into Dictyostelium cell pellet isshown. All results are expressed as control at 60 minutes. Insets showdose response curves. Statistics and dose response curves werecalculated using Graphpad Prizm™ software. All data are replicates of atleast 3 independent experiments and show mean±SEM.

FIG. 6 demonstrates that VPA induces endocytosis independent lipiddroplet accumulation of bodipy in Dictyostelium. (a) Images of bodipyfatty acid accumulation in Dictyostelium in the absence (i) or presence(ii) of 0.5 mM VPA. VPA significantly increased the droplet intensityand average diameter of lipid droplets compared to control (b) (t test,*** p<0.001, * p<0.05). The actin polymerising inhibitor latrunculin (10μM) did not completely inhibit VPA induced increase in ³H arachidonicacid uptake. All data are replicates of at least 3 independentexperiments and show mean±SEM.

FIG. 7 demonstrates that knockout PlaA cells are protected fromVPA-induced inhibition of development. (A) Alignment of PlaA proteinsequence from Dictyostelium discoideum (XP_642421.1) and an iPLA₂protein sequence from Homo sapiens (AAD08847). Sequences show conservedhomology at ATP binding and lipase sites. Alignment was carried outusing BLAST software. (B) Radiolabel release from wild type and PlaAcells show a similar reduction of radiolabel release in the presence ofVPA. (C) Development images of Dictyostelium wild type (Ax2) cells orPlaA-ve cells at 30 hours in the absence or presence of 1 mM VPA (asindicated). Scale bars represent 500 μm.

FIG. 8 demonstrates that PLA₂ inhibitors phenocopy VPA-induced ³Hrelease from fatty acid labelled cells. (A) Aggregation competentDictyostelium wild type (Ax2) cells were pre-incubated with ³H AA andthe release of ³H into external buffer is shown in the presence/absenceof PLA₂ inhibitors. Inhibitor x=80 μM BEL, a Ca²⁺ PLA₂ inhibitor, y=20μM BPB, a general PLA₂ inhibitor and z=50 μM MAFP, a Ca²⁺ dependent andCa²⁺ independent cytosolic PLA₂ inhibitor. Mix=combination of allinhibitors. (B) PLA₂ inhibitors do not mimic VPA-dependent fatty aciduptake. Cells were incubated in the presence of VPA and PLA₂ inhibitormix (see methods). Uptake of ³H into Dictyostelium cell pellet is shown.(C) VPA induced ³H arachidonic acid uptake is independent of CoAactivation. Quantification of CoA activated palmitic acid in wild type,wild type+0.5 mM VPA or fcsA −/− Dictyostelium (one way ANOVA, Dunnet'spost hoc *** p<0.001). (D) Uptake of ³H arachidonic acid in wild typefcsA −/− or fcsB —/− cells in the absence or presence of 0.5 mM VPA (oneway ANOVA, Bonferroni post hoc, *** p<0.001).

FIG. 9 shows the effect of VPA analogues on ³H arachidonic acid uptakeand release. VPA induced parallel enhanced uptake and reduced release ofradiolabel, effects which were enhanced by 2-isopropyl pentanoic acid(PIA) and reduced by 4-methyloctanoic acid. PLA₂ inhibitor cocktailinhibited both the uptake and the release of radiolabel (one way ANOVA,Bonferroni post hoc, ns not significant, *** p<0.001).

1. Materials & Methods

1.1 Chemicals and Mutants

All chemicals were provided by Sigma UK Ltd. Valproic acid congenerswere provided by Sigma Aldrich UK, Alfa Aesar/Avocado, ChemSampCo,ChemCo, The NCI/DTP Open Chemical Repository (http://dtp.cancer.gov) orTCI europe. Radiolabelled ATP was provided by Perkin Elmer Ltd.Dictyostelium mutants were provided by Dictybase.org, apart from thequintuple PI3K (and PTEN) knockout strain and the rPKA knockout kindlyprovided by R.Kay (Cambridge, UK) and A.Noegel (Koeln, Germany).Dictyostelium media (Axenic) was supplied by Formedium (Norfolk, UK). ³Harachidonic acid purchased from Hartmann analytic (Germany) and ³Hpalmitic acid from Perkin Elmer (Cambridge, UK).

1.2 Cells and Development

Dictyostelium cells were grown in Axenic medium or on Sussmans mediaplates in association with Raoultella planticola (Drancourt et al.,2001). All cell labelling used cells shaking (120 rpm) at 22° C., andcells were artificially developed by pulsing with cAMP (25 nM finalconcentration) every 6 min for 4 hours at 2.5×10⁶ cells/ml in phosphatebuffer (16.5 mM KH₂PO₄, 3.8 mM K₂HPO₄ pH 6.2) as described previously(Boeckeler et al., 2006).

1.3 Dictyostelium Phospholipid Labelling and Inositol Analysis

A saponin-based cell permeabilization protocol for Dictyostelium wasadapted for these experiments (Pawolleck & Williams, 2009).Dictyostelium AX2 cells were developed for 5 hours as previouslydescribed (Boeckeler et al., 2006) (pulsed with cAMP to achieve finalconcentration of 25 nM), transferred to still dishes (2.5 cm), allowedto settle to give a confluent monolayer in KK2 (20 mM potassiumphosphate buffer, pH 6.1) and pre-treated with compound (0.5 mM VPA orrelated compound or 50 μM LY294002) for 3 min. At regular timeintervals, buffer was replaced with labelling solution (139 mM sodiumglutamate, 5 mM glucose, 5 mM EDTA, 20 mM PIPES pH 6.6, 1 mM MgSO4.2H2O,0.25% (w/v) saponin, 1× phosphatase inhibitor cocktails 1 and 2 (RocheLtd), and 1 μCi/ml γ[32P]ATP) supplemented with compounds at definedconcentrations.

Following a 6 min incubation, labelling solution was removed and cellswere lysed in acidified methanol and phospholipids were separated aspreviously detailed (Williams et al., 1999). Phospholipid labelling wasquantified using a Typhoon phosphor-imager. Even loading was determinedusing total lipid stain with copper sulphate. Inositol levels weremeasured from five hour developed cells (similar to phospholipidlabelling), following lyophilisation, as previously described (Maslanski& Busa, 1990).

1.4 In Vitro Epilepsy Model

The rats (p21) were decapitated after killing by intraperitonealinjection with an overdose of pentobarbitone (500 mg/kg). The brain wasremoved and placed in ice-cold sucrose solution in mM: NaCl 87, KCl 2.5,MgCl₂ 7, CaCl2 0.5, NaH₂PO₄ 1.25, sucrose 75, glucose 25, equilibratedwith 95% O₂/5% CO2 (pH 7.4). Horizontal combined entorhinalcortex-hippocampus slices (350 μm) were prepared with a Leica vibratome(Leica VT1200S) and were then stored in an interface chamber thatcontained artificial cerebrospinal fluid solution (aCSF) containing inmM: NaCl 119, KCl 2.5, MgSO₄ 4, CaCl2 4, NaHCO₃ 26.2, NaH₂PO₄ 1, glucose11, and gassed with 95% O₂/5% CO₂. They were stored for over one hourbefore being transferred to a submersion recording chamber continuallyperfused with carbogenated aCSF for recording. Field potentialrecordings were made by placing glass microelectrodes (˜1-2 MΩ) filledwith aCSF solution in stratum radiatum of CA1. Bipolar stimulatingelectrodes were positioned in the Schaffer collateral/commissural fibrepathway in stratum radiatum to confirm slice viability. In the PTZ acuteseizure model, PTZ (2 mM) was added to the perfusate and [K+] wasincreased to 6 mM in order to induce epileptiform activity (Armand etal., 1998). In the low Mg²⁺ acute seizure model, Mg²⁺ free aCSF wasapplied to generate rhythmic short recurrent discharges. Novelanticonvulsants were applied once the frequency and amplitude of theepileptiform discharges were stable over a period of 10 min.Anticonvulsant effects were evaluated by measuring the variation offrequency of the discharges every minute. The data acquired from the 30to 40 minutes after application novel anticonvulsants were compared byANOVA followed by post-hoc testing using Tukey test, using SPSSstatistical analysis.

1.5 In Vivo Status Epilepticus

This method has been described in detail previously (Walker et al.,1999). In brief, male Sprague Dawley rats (300-400 mg) were anesthetizedwith 1-2% isoflurane in O₂. An earth electrode was positionedsubcutaneously, and a monopolar recording electrode was implantedstereotactically into the right hippocampus (coordinates, 2.5 mm lateraland 4 mm caudal from bregma). A bipolar stimulating electrode wasimplanted in the right hemisphere and advanced into the angular bundle(coordinates, 4.4 mm lateral and 8.1 mm caudal from bregma) to stimulatethe perforant path. The depths of the electrodes were adjusted tomaximize the slope of the dentate granule cell field potential (Guo etal., 1999). The electrodes were held in place with dental acrylic andskull screws. The animals were allowed to recover from anaesthesia.Seven days later, the perforant path was electrically stimulated with4-5 mA 50 μsec monopolar pulses at 20 Hz for 2 hr; this inducedself-sustaining status epilepticus. After 10 min of self-sustainingstatus epilepticus, compounds or vehicle were administered and thebehavioural seizures and EEG were monitored for 3 hours. At this pointdiazepam (10 mg/kg) was administered to all animals to stop the statusepilepticus. Groups were compared by ANOVA followed by post-hoc testingusing Tukey test, using SPSS.

1.6 Fatty Acid Uptake and Release

Dictyostelium cells were labelled with tritiated fatty acid in shakingliquid culture at 1.5×10⁶ cells/ml with 0.5 μCi of ³H labelled fattyacid added in 0.5% BSA (fatty acid free BSA) per 2 treatments. Sampleswere taken at indicated times by removing 4.5×10⁶ cells, washing once inphosphate buffer and re-suspending in phosphate buffer prior toscintillation counting. For fatty acid release experiments, cells werepulsed (as above) for 4 hours, and cells were resuspended in phosphatebuffer with fatty acid free BSA (0.5%) at 1.5×10⁶ cells/ml and timepoints were taken over one hour. Cells (4.5×10⁶ per time point) werewashed to remove unincorporated radioactivity and at indicated times andthe supernatant was analysed via scintillation counting. Modellingresults employed using Graphpad Prism software. Bodipy labellingemployed 4 hour pulsed cells, incubated with fluorescent fatty acid(Invitrogen) for 30 min in the presence or absence of VPA (0.5 mM) andimages were recorded on an Olympus IX 71 inverted fluorescencemicroscope with Retiga FastA 1394 camera and analysed by ImagePro™software.

1.7 Mutant Isolation and Recapitulation and Development

Screening of a REMI library was carried out as previously described(Kuspa & Loomis, 2006) using Ax2 background, with VPA resistant mutantsselected for the ability to develop in the presence of 1 mM VPA on R.planticola. Identification of the ablated gene, enabled the identifiedPLAa (DDB_G0278525) to be recapitulated using by homologousrecombination of a knockout cassette. Primers used for amplifying regionwithin the open reading frame of the gene were (5′ATGGGAGATAATAAAAAAGAAAATATCAG and 3′ TAAGAATTCATGGGAGATAA TAAAAAAGAAAATATCAG, cloned by pCR2.1 TOPO (Invitrogen Ltd)), cloned intopUC19 using EcoR1 digestion, and SmaI digested fragment from pBLPblp(Faix et al, 2004) was inserted into the EcoRV site of the insert.Genetic ablation was confirmed by PCR analysis. Developmental resistanceto VPA was assessed by plating cells (1×10⁶) on 47 mm nitrocellulosefilters (Millipore) soaked in phosphate buffer containing either 1 mMVPA or control, and development was recorded after 30 h unless otherwisestated. Development images were observed using a Leica CLS 150×microscope and images recorded using QICAM FAST 1394 camera. Fatty acidactivation was determined by the method established by Wilson et al.(Wilson et al, 1982) with slight modifications. Briefly, extracts wereprepared by sedimentation of 1×10⁷ axenically grown cells, washing themonce in 10 ml precooled 1 M Tris-HCl (pH 7.5) and lysing them for 30 minin 100 μl 1 M Tris-HCl (pH 7.5) containing 1% Triton and ProteaseInhibitor Cocktail (P8340, Sigma-Aldrich, Germany) at 4° C. Twenty mg ofprotein extract in a volume of 140 ml were diluted into 400 μl of abuffer containing 250 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 3 mM ATP, 0.6mM EDTA, 0.25% Triton, and 2.5 mM DTT. 40 μl of unlabelled palmitic acid(P9767, Sigma-Aldrich, Germany) from a 100 μM methanol stock and 5 μl3H-palmitic acid (20 μM, 1 mCi/ml) served as substrates. The reactionwas started by addition of 20 μl 10 mM coenzymeA-solution and incubatedat 35° C. To stop the reaction 500 μl Dole's medium (0.4 ml isopropanol,0.1 ml n-heptane, 10 μl H₂SO₄) was added after 10 min. Separation of thephases was achieved by centrifugation for 30 sec at 14,000 rpm in atabletop centrifuge. The organic phase was discarded and the aqueousphase was washed six times with 300 μl of n-heptane to removenon-activated fatty acids before the radioactivity of the acyl-CoAthioester remaining in the aqueous phase was determined in 2 ml ofLumasafe™ Plus fluid (Lumac LSC, Groningen, The Netherlands) in ascintillation counter.

2. Results

2.1 VPA Attenuates Phosphoinositide Signalling

Since the acute effect of VPA on phosphoinositide signalling has notbeen characterised, the inventors firstly examined the time- andconcentration-dependence of drug action in Dictyostelium (FIG. 1). Arapid reduction in phosphoinositide phosphorylation was seen following0.5 mM VPA treatment, with a 49% reduction in both radio-labelled PIPand PIP₂ turnover during 6 min VPA treatment, increasing to 94%reduction in the turnover of each phosphoinositide compound following 60min treatment (FIG. 1a ). These values indicate a combination ofphosphoinositide synthesis and degradation within the cell, thus reflectphosphoinositide turnover, although phosphatase activity is blockedusing an inhibitor cocktail. The acute nature of this effect occurs witha similar speed to that of seizure control following intravenous VPAinjection in a mouse seizure model (Honack & Loscher, 1992). Theattenuation of phosphoinositide signalling was also concentrationdependent, with a 25% and 42% inhibition of PIP and PIP₂ turnoverrespectively at 0.25 mM VPA following 9 min treatment increasing to a68% and 79% reduction at 0.5 mM VPA respectively (FIG. 1b )—theseconcentrations are found in the therapeutic use of VPA (0.4-0.7 mM inplasma) (DSM IV, 2000). Under these conditions, this inhibitory effectof VPA provides an EC50 of 154 μM, and is independent of uptake (sincecells are permeabilized with saponin). The acute, strong inhibition ofphosphoinositide signalling caused by VPA made this effect of potentialtherapeutic interest.

A rapid reduction in phosphoinositide phosphorylation was initiallythought to occur through inhibition of an unidentified lipid kinaseactivity. Analysis of lipid kinases traditionally employspharmacological inhibition with enzyme class-specific compounds, butthese studies are complex due to the large number ofphosphatidylinositol kinase enzymes and overlapping effects betweeninhibitors. Since previous studies have suggested a role for VPA inattenuating the phosphatidylinositol 3-kinase (PI3K) signalling pathway(Xu et al., 2007), the inventors analysed the effect of ablating fivedifferent type 1 PI3K genes in a single cell line (Hoeller & Kay, 2007)on phosphoinositide signalling (FIG. 2a ). These cells showed a 28% and44% reduction in the formation of PIP and PIP₂ production respectivelycompared to wild type cells, suggesting a major role of these enzymes inphosphoinositide signalling. To test for a PI3K-dependence of theVPA-catalysed phosphoinositide reduction, VPA (0.5 mM) was added tothese cells. It was found that VPA reduced PIP and PIP₂ production by48% and 70%, respectively compared to untreated cells following 9 mintreatment (FIG. 2b ), indicating that these five ablated enzymes are notthe target of VPA in attenuating phosphoinositide turnover.

To investigate a role of other lipid kinases in VPA-catalysedphosphoinositide attenuation, the inventors analysed two othernon-related phosphatidylinositol kinases: the rPKA knockout mutantlacking an endosomal G-protein-coupled receptor protein containing aphosphatidylinositol 5 kinase (PIPSK) domain (Bakthavatsalam et al.,2006); and the PIPKinA mutant that lacks a nuclear phosphatidylinositol4/5 kinase activity (Guo et al., 2001). Ablation of rPKA (FIG. 2a )showed a 30% and 54% reduction in PIP and PIP₂ production, respectivelycompared to wild type cells, with VPA treatment causing an additional72% and 33% reduction compared to untreated cells (FIG. 2c ). Ablationof PIPKinA showed no significant change in PIP and PIP₂ production (FIG.2a ), with VPA treatment causing a 54% and 68% reduction in PIP and PIP₂synthesis respectively compared to untreated cells (FIG. 2d ). All threecell lines were still sensitive to pharmacological inhibition of PI3Kactivity (using 50 μM LY294006—an inhibitor of PI3K activity),confirming these variations were related to attenuated phosphoinositideturnover (FIGS. 2b-d ). The reduced sensitivity of all three lipidkinase mutants suggests a common mechanism of VPA action independent ofspecific phosphatidylinositol kinase action.

Since another mechanism for regulating phosphoinositide signalling isthe recycling of phosphatidylinositol, via inositol phosphates (FIG. 2e), the inventors analysed phosphoinositide turnover in isogenic mutantswith this recycling pathway blocked or activated. Cells lacking thesingle phospholipase C gene (Drayer et al., 1994) showed no significantreduction in PIP and PIP₂ turnover compared to wild type cells (FIG. 2f), and showed a VPA-catalysed reduction in PIP and PIP₂ signalling closeto that for wild-type cells (73 and 75% for PIP and PIP₂ respectively).Cells with approximately three-fold higher inositol trisphosphate(InsP₃) caused by prolyl oligopeptidase ablation (PO; Williams et al.,1999, Williams et al., 2002) showed a slight decrease in PIP levels inuntreated cells (and no significant change in PIP₂ levels) and aVPA-catalysed reduction in PIP and PIP₂ signalling by 66% and 68%respectively. Furthermore, the inventors have previously shown thatinhibition of inositol monophosphatase (IMPase) activity by 10 mMlithium does not attenuate phosphoinositide signalling following acute(9 min) treatment (King et al., 2009), however extended lithiumtreatment (60 min) reduces PIP and PIP₂ levels, and this effect isovercome by over-expression of IMPase (King et al., 2009). Incomparison, over-expressing IMPase did not overcome extended VPAtreatment (60 min; 0.5 mM) (FIG. 2g ). These results suggest thatelevating or reducing recycling of inositol through inositol phosphatesignalling does not play a major regulatory role in acutephosphoinositide production and does not overcome VPA-catalysed acutereduction in phosphoinositide signalling in this model. These resultsthus provide the first strong evidence for a mechanism of action ofVPA—independent of inositol depletion—in targeting phosphoinositidesignalling.

2.2 Identifying Novel Compounds Showing Increased PhosphoinositideAttenuation

The identification of an acute effect of VPA in attenuatingphosphoinositide signalling enabled the investigation of the structuralrequirements for this effect. The effect of compounds tested for thepresent invention on phosphoinositide attenuation are summarised inTable 1 below.

TABLE 1 PIP Chemical Chemical Chemical Level (% category (common name)(IUPAC nomenclature) control) SD valproic acid (VPA) 2-propylpentanoicacid 32.0 8.7 Shorter than 5 carbons backbone (the longest aliphaticside chain) acids Isovaleric Acid 3-methylbutanoic acid 37 93-methylbutanoic 99.1 11.4 GABA 4-aminobutanoic acid 60.8 3.8 TBAtert-butylacetic acid 21.4 4.1 PIA propylisopropylacetic acid 15.4 2.2DIA diisopropylacetic acid 18.6 4.5 5 carbon backbone acids4-methylpentanoic acid 60 11.8 2-methyl-2-pentenoic acid 14.8 3.84-methyl-2-pentenoic acid 54.8 14.1 2,4-dimethyl-2-pentenoic acid 38.46.8 trans-pent-2-enoic acid 50.4 9.1 2-methylpentanoic acid 59 7.33-methylpentanoic acid 64 13.6 4-methyl-2-pentenoic acid 121 252,4-dimethyl-2-pentenoic acid 94 14 3-methylvaleric acid3-methylpentanoic acid 66 12 4-methylvaleric acid 4-methylpentanioc acid102 18 3-methylpentanoic acid 67 16 2,2-dimethyl-4-pentenoic acid 60 73-methyl-4-pentenoic acid 84 6 6 Carbon backbone acids 4-methylhexanoicacid 68 3 2-methylhexanoic acid 68.1 14.8 5-methylhexanoic acid 7.2 0.72-ethylhexanoic acid 22.2 5.3 2,2-dimethylhexanoic 29.7 9.73,5,5-trimethylhexanoic acid 32 13 4-hexenoic acid, (cis + trans) 58 157-9 carbon backbone acids 2-methylheptanoic 16.1 6.4 4-methyloctanoicacid 12 1.3 4-ethyloctanoic acid 13.2 1.8 4-methylnonanoic acid 45 16 11carbon backbone acids 3-methylundecanoic acid 50 0 Straight-chain acidsvaleric acid pentanoic acid 66.9 7.8 n-caproic acid hexanoic acid 49.210.1 enanthoic acid heptanoic acid 31.2 5 caprylic acid octanoic acid16.7 3.4 pelagonic acid nonanoic acid 8 1.7 capric acid decanoic acid12.9 1.4 Lauric acid dodecanoic acid 42 3 Margaric acid heptadecanoicacid 92.3 2.8 Other acids Diphenylacetic acid 2,2-diphenylacetic acid 3911 TMCA tetramethylcyclopropane 33.9 7.7 carboxylic acid Derivatizedcarboxylic acids (amides) valpromide (VPD) 2-propylpentamide 69.3 13.3valnocatmide (VCD) 2-ethyl-3-methyl valeramide 64 2.9 TMCDtetramethylcyclopropane- 72.5 7.2 carboxamide MTMCDN-methyl-tetramethyl- 50.8 6.9 -cyclopropane carboxamide PIDpropylisopropylacetamide 59 12.2 Tert-butyl amide 47.6 12 n-propyl2-methylvalerate 121 13.6 Aldehydes methylvalerate 46 5.5 valeraldehydepentanal 52 10.8 octanal 260 199 nonanal 99 13 Alcohols2-propyl-1-pentanol 101 13 2-butyl-1-octanol 93 53 2-hexyl-1-decanol 19138

Although the majority of compounds analysed showed some inhibitoryeffect on phosphoinositide turnover (Table 1), a number of structuresshowed greater phosphoinositide signalling inhibition than VPA. Thesehighly active compounds fit into two structural groups: the firstcomprising branched fatty acids with a roughly similar structure to VPA;and a second novel group of compounds with or without short side chainsin various positions on the backbone. Within this latter group, fattyacids show a strong dependence on length, whereby 8-10 carbon backboneacids are highly active (e.g. 4-methyloctanoic acid reduces PIP and PIP₂signalling by 88% and 93% respectively, and nonanoic acid by 92% and 93%respectively; Table 1) and increased or decreased backbone lengthreduces activity. All highly active compounds in this group are fattyacids, without predicted teratogenicity (Eickholt et al., 2005) and showa positive association with lipophilicity. This effect is alsoindependent of acidic function, since variable activity is shown withstraight carbon acids of equivalent acidity (pKa, Table 2 which shows acomparison of phosphoinositide attenuation and pKA values for VPA andstraight chain acids in Dictyostelium). These structural distinctionsprovide the first characterization of VPA congeners for this effect ofphosphoinositide attenuation. Interestingly, high structural specificityhas previously been show for fatty acids in both anticonvulsant as wellas antiallodynic (anti-neuropathic pain) activities (Kaufmann et al.,2009). Preliminary observation of behaviour in animal models for onerelated compound does not suggest a strong sedative effect.

TABLE 2 Total number of PIP level Acid Carbon atoms pKa % control SD VPA8 4.6 32.0 8.7 Pentanoic acid 5 4.84 66.9 7.8 Hexanoic acid 6 4.85 49.210.1 Heptanoic acid 7 4.89 31.2 5 Octanoic acid 8 4.89 16.7 3.4 Nonanoicacid 9 4.95 8 1.7 Decanoic acid 10 4.90 12.9 1.4 dodecanoic acid: 114.85 42 3 heptadecanoic acid 17 4.78 92.3 2.8

VPA has been identified as an inhibitor of de novo inositolbiosynthesis, indirectly blocking the production of inositol-1-phosphatefrom glucose-6-phosphate (Shaltiel et al., 2004, Shaltiel et al., 2007a,Vaden et al., 2001). A role for VPA-attenuation of inositol signallinghas been widely shown in models ranging from yeast (Vaden et al., 2001)and Dictyostelium (Williams et al., 1999, Williams 2002) toCaenorhabditis elegans (Tokuoka et al., 2008), rats and humans (Shaltielet al., 2007a, Shaltiel et al., 2007b). Measurement of inositol andinositol trisphosphate (InsP3) levels and the inositol-dependentspreading of mammalian growth cones have all been used to show inositoldepletion. Since it is not clear if a VPA-induced reduction in inositollevels may cause the a cute reduction in phosphoinositide signallingshown here, the inventors analysed phosphoinositide turnover usingVPA-related compounds shown to be active in inositol depletion.Compounds showing strong InsP3 depletion in Dictyostelium withconcomitant inositol-depletion dependent enlargement in mammalian growthcones include 2-methyl-2-pentenoic acid (Eickholt et al., 2005) and thiscompound showed stronger phosphoinositide attenuation than VPA.Interestingly, substituting the carboxylic acid moiety of compoundsshowing high phosphoinositide attenuation (VPA) with a carboxamide group(yielding the corresponding amide (VPD) reduces the inhibitory effect onphosphoinositide turnover and reduces growth cone spreading, and VPDshows weak inhibition of human myo-inositol synthase proposed as theVPA-target in inositol depletion (MIP synthase (Shaltiel et al., 2004,Shaltiel et al., 2007a)). These inositol-depleting andphosphoinositide-attenuating compounds are found mainly within the firststructural group of compounds (described above), and also contain anumber of potent anticonvulsants second generation to VPA currentlyunder investigation (Bialer & Yagen, 2007). None of the novel family oflonger backbone compounds identified in this study have been analysed ininositol depletion studies.

Since reduction in the inositol levels may provide the mechanism ofthese compounds in phosphoinositide attenuation, the inventors analysedinositol levels in treated Dictyostelium cells using a range ofcompounds from both structural groups showing variable phosphoinositideattenuation). In these experiments, VPA gave no significant reduction ininositol levels in the time period shown to cause phosphoinositideattenuation (9 min), nor did any other compound tested, and thus nocorrelation was found between phosphoinositide attenuation and inositoldepletion. This conclusion is in agreement with previous data, based inDictyostelium, showing the acute inhibition of inositol monophosphatase(by lithium) does not give rise to phosphoinositide attenuation (King etal., 2009), and depletion of inositol trisphosphate in this model by VPArequires 6 hour treatment (Williams et al., 1999)—considerably longerthan the time periods used here. These experiments therefore suggestphosphoinositide attenuation provides a novel effect of VPA inDictyostelium and identifies a range of compounds showing increasedefficacy for this effect. Since increased PIP and PIP₂ levels have beenobserved during seizures in animal models (Van Rooijen et al., 1986),and the inventors have discovered a novel family of compounds causingthis effect.

2.3 Novel Compounds Show Enhanced Efficacy in In Vitro EpileptiformModels

Since it is not possible to repeat these radio-labelling experiments inin vivo animal systems to replicate the inventors' mechanism-dependentfindings in higher models, the inventors instead analysed the efficacyof the novel family of compounds in seizure control. For theseexperiments, they employed a VPA-sensitive pentelenetetrazol (PTZ) invitro model of epileptiform activity (Armand et al., 1998) to analysethree compounds from the novel family (FIGS. 3a,b ) with an eight carbonbackbone with variable side chain position and length. VPA significantlydecreased the frequency of epileptiform discharges (Armand et al., 1998;FIG. 3b ; VPA: 75.1±1.7□). the application of equimolar concentrationsof each eight carbon backbone compound also strongly reduced dischargeswith a significantly greater efficacy than VPA (FIGS. 3b,c ).Application of all three novel compounds greatly reduced seizuredischarge frequency (4-methyloctanoic acid, 49.1±4.4%, 2-propyloctanoicacid is 5.3±3.3 and 2-butyloctanoic acid 5.2±5.0% all P=0.005 comparedto VPA). The inventors also extended these compounds to show a similarefficacy for straight chain nine- and ten-carbon backbones (nonanoicacid, 20.9±7.50, P=2×10-6 compared to VPA; decanoic acid 0.23±0.23%,P=2×10-6 compared to VPA FIGS. 3d, e ). This activity was not seen withshorter backbones (e.g. 5 carbon pentanoic acid—data not shown). Thesehighly potent compounds have not previously been associated with seizurecontrol, and would not be predicted to show teratogenic effects (Guo etal., 1999).

To show that the effect of these compounds was not seizure-modelspecific, the inventors further investigated the effect of one of thesecompounds in the in vitro low Mg²⁺ seizure model (FIGS. 3 f, g, h). Theinventors chose 4-methyloctanoic acid (hircinoic acid), since this isendogenous to animal systems (Johnson et al., 1977). VPA (1 mM) weaklyreduced the frequency of recurrent short discharges in this model.Application of 4-methyloctanoic acid almost abolished the frequency ofrecurrent short discharges 30-40 minutes after application. These datatherefore suggest that 4-methyloctanoic acid shows enhanced activityover VPA in multiple in vitro models of epileptiform activity.

2.4 Novel Compounds Show Enhanced Status Epilepticus Control

To demonstrate further efficacy in animal seizure models with thesecompounds, the inventors tested 4-methyloctanoic acid in an in vivomodel of status epilepticus (FIG. 4). For this test, status epilepticuswas induced by stimulation of the perforant path in awake, freely movingrats as has been previously described (Holtkamp et al., 2001, Walker etal., 1999; FIG. 4a ). The inventors have previously found that VPA iseffective in this model at high dose (600 mg/kg) but has only partialeffectiveness at a lower dose (400 mg/kg). The inventors thereforecompared the efficacy of 4-methyloctanoic acid (400 mg/kg) against VPA(400 mg/kg). 4-methyloctanoic acid has a marginally higher molecularweight (MW=158) than that of VPA (MW=144) and so this dose represents aslightly lower molar dose of 4-methyloctanoic acid. VPA stronglyattenuated seizures in this model 2 hours after treatment, with reducedefficacy three hours post treatment (FIG. 4b ), whereas 4-methyloctanoicacid protected against seizures over the test period. Both compoundsreduce spike amplitude and frequency (FIG. 4): VPA reduced spikeamplitude (75.2±9.2% in the first hour; 61.1±8.3% in the second hour;55.1±6.6% in the third hour) (FIGS. 4e,f ). In comparison,4-methyloctanoic showed significantly better control, reducing the meanspike frequency to 39.3±11.3% in the first hour (significantly betterthan VPA p<0.05); 18.1±10.3% in the second hour (p<0.05 compared to VPA)and 26.9±12.3% in the third hour (FIGS. 4e,f ). 4-methyloctanoicterminated status epilepticus (defined as a spike frequency of less than1 Hz) in all status epilepticus animals. Furthermore, 4-methyloctanoicacid completely stopped the seizures in all 7 animals after 2 hours,whilst VPA decreased seizure severity but did not terminate the seizuresin any (P=0.0003, Fisher's exact test), and this effect was maintainedin five out of seven animals given 4-methyloctanoic acid by three hours(P=0.01, Fisher's exact test).

2.5 VPA Regulates Fatty Acid Uptake and Release

To analyse a role for VPA-mediated regulation of arachidonic acidrelease in Dictyostelium, the inventors developed an assay based uponthe release of radiolabel from cells containing tritiated fatty acidover time. Using this assay, the inventors showed that following ³H-AAlabelling of cells, the release of radiolabel into media was linear overa 45 min period (FIG. 5A). The effect of VPA on radiolabel release fromAA-labelled cells was acute and dose dependent, whereby VPA induced adecrease in the release with an IC50 of 89 μM. This effect was notspecific to AA, since release of tritiated palmitic acid was alsoinhibited in the presence of VPA with an IC50 of 163 μM (FIG. 5C). Theacute nature of this effect is seen with a significant inhibitionfollowing 30 min exposure (p<0.05).

Since reduced release of labelled fatty acid may be due to itsre-incorporation into lipids, the inventors also measured fatty aciduptake by measuring radiolabel incorporation of fatty acids into cells.Like fatty acid release, incorporation of tritiated AA was linear over a30 min period (FIG. 5B), however, VPA caused a dose-dependent increasein the uptake of AA, with an EC50 of 47 μM. This effect was also seenusing palmitic acid, with an EC50 value of 160 μM (FIG. 5D) and wassignificant following 30 min drug treatment (p<0.05).

In order to test if the above effects occur through simple fatty acidmembrane insertion, the inventors visualised fatty acid uptake using acompound containing a 12 carbon fatty acid chain linked to fluorescenthead group (bodipy; FIG. 6A) (Worsfold et al., 2004). Upon incubation ofcells and bodipy-labelled lipid, 0.5 mM VPA caused an increase in theintensity and diameter of fluorescent lipid droplets within cellscompared to untreated cells (FIG. 6B). This results show that VPA alsoincreased the uptake of this fatty acid, and that the drug increasedfatty acid storage within lipid droplets.

2.6 VPA-induced Fatty Acid Uptake Occurs Independently of Actin Dynamics

Uptake (and release) of compounds in Dictyostelium is likely to beregulated by cellular mechanisms controlling macropinocytosis, thuschanges in fatty acid incorporation may be due to simple regulation ofthis process. To examine this, and since macropinocytosis is dependentupon actin polymerisation, the inventors used latrunculin (10 μM), aninhibitor of actin polymerisation (de Oliveira and Mantovani, 1988), toobserve the effects on fatty acid uptake. Inhibition of actinpolymerisation decreased uptake in control cells. However, latrunculinfailed to attenuate VPA-induced AA uptake suggesting VPA-induced fattyacid regulation was independent of vesicle dynamics (FIG. 6C).

2.7 Genetic Ablation of PLA₂ Activity does not Reverse Fatty AcidPerturbation

To identify specific genes controlling the effect of VPA in this model,the inventors carried out a restriction enzyme mediated integration(REMI) mutant screen to identify loci controlling the effect of VPAduring development (Kuspa and Loomis, 2006). VPA inhibits thedevelopment of Dictyostelium at concentrations found in plasma ofpatients receiving VPA treatment (0.28-0.7 mM; (Silva et al., 2008))(FIG. 7B). One mutant isolated in this screen contained an ablated PLA₂gene (van Haastert et al., 2007), with the encoded protein showingsimilarity to Ca²⁺-independent enzymes, and containing conserved ATPaseand lipase motifs (FIG. 7A), and the mutant showed partial resistance toVPA during development (FIG. 7B). However, the knockout cell line didnot attenuate radio-label release from cells (FIG. 7C) suggesting thatalthough disruption of the PLA₂ gene offered partial protection to VPAduring development, it was not enough to prevent gross VPA-induced fattyacid release.

2.8 VPA Regulation of Fatty Acid Signalling is not Phenocopied by PLA₂Inhibition

Since VPA has been suggested to regulate phospholipase A2 (PLA₂) relatedsignalling (Rao et al., 2008), and since ablation of a single PLA₂ geneprovided only partial resistance to VPA during development and no effecton gross radiolabel release or fatty acid uptake (FIG. 7), the inventorsassessed the role of pharmacological inhibition of PLA₂ on AAregulation. Chemical inhibitors of different PLA₂ class specificity (BEL[80 μM], a Ca²⁺-independent PLA₂ inhibitor (Ackermann et al., 1995);MAFP [50 μM], a Ca²⁺-dependent and Ca²⁺-independent cytosolic PLA₂inhibitor (Balsinde and Dennis, 1996, Lio et al., 1996); and BPB [20μM], a phospholipase A2 inhibitor (Mitchell et al., 1976) all reducedradiolabel release from ³H-AA cells in a similar manner to VPA treatment(FIG. 8). Differing specificity for these inhibitors was shown since acocktail of all three inhibitors provided a cumulative inhibition ofrelease. In contrast to the effect of VPA on release (causing anincrease in fatty acid over time), chemical inhibition of PLA₂ activitycaused a reduced uptake of fatty acid (FIG. 8). This data suggests thatPLA₂ inhibition partially phenocopies the effect of VPA in modifying AAsignalling, but that VPA has a more generalised effect on fatty acidsignalling.

2.9 VPA-Induced Fatty Acid Uptake is not Dependent on Fatty AcidActivation

To test whether the incorporation of fatty acids was dependent onactivation, the inventors firstly tested the ability of cell extracts toactivate palmitic acid to form PaA-CoA. In these experiments, incubationof cell extracts with ³H-PaA and coenzyme A enabled the activation ofthe fatty acid that was subsequently separated by differential solventsolubility and quantified (von Lohneysen et al., 2003). Inclusion of VPA(1.0 mM) either with cell extracts during the activation assay, or bypre-treatment of cells prior to preparation of extracts (10 min, 1 mM)had no effect on fatty acid activation, whereas ablation of theperoxisomal fatty acid CoA synthase A enzyme (FcsA)—the enzymeresponsible for fatty acid CoA activation in endosome—showed asignificant reduction in PaA-CoA synthesis (FIG. 8) compared towild-type cells. Furthermore, cell lines lacking fcsA (DDB_G0269242;(von Lohneysen et al., 2003)) showed a VPA-induced increase in fattyacid uptake in a similar manner to wild type cells (FIG. 8). Theseresults suggest that the effect of VPA in regulating fatty acidsignalling is independent of fatty acid CoA activation as was previouslysuggested (Bazinet et al., 2006b).

2.10 Structural Specificity of Induced Fatty Acid Release

SAR studies identify the physical requirements for compounds to cause aneffect, and these studies can help to distinguish between discreteeffects of a compound. To examine the structural dependency of VPA onradiolabel release following ³H-AA incorporation, the inventors employeda range of compounds related to VPA with varying carbon backbone andside chain lengths, head group, enantiomeric specificity and saturationand measured release following 30 min 0.5 mM treatment. The resultsobtained are summarised in Table 3 below.

TABLE 3 AA release [% Compound structure of control] Control 100  VPA(2-propylpentanoic acid)

50 2-propyloctanoic acid

45 2-propyldecanoic acid

56 decanoic acid

62 4-methylnonanoic acid

63 nonanoic Acid

66 4-methyloctanoic acid 66 octanoic acid

72 2-ethyldecanoic

73 4-ethyloctanoic acid

87 dodecanoic acid

89

The compounds tested showed a range of inhibitory activity, with highstructural specificity, and the strength of activity was independent ofacidity and lipophilicity (pKA and log P values respectively).

Since VPA gave a reduction in activity to 50% of control, the inventorsdefined compounds, such as 2-propyloctanoic acid, as highly active sincethey reduce activity to 45% or below. These highly active compounds werecarboxylic acids (since valpromide gave virtually no inhibition, datanot shown) branched at the second carbon, with the most active compoundscontaining an isopropyl group. Unlike teratogenicity (Eickholt et al2006), a tertiary-substituted C2 still showed activity, and long- andmedium-length straight chain fatty acids were still active. Branchedcompounds were stronger than corresponding straight chains, with apreference for longer side chains (propyl- giving stronger inhibitionthat methyl- side chains). Finally, unsaturated compounds showed areduction in inhibitory activity. This SAR study represents a noveldescription of a VPA-catalysed effect.

To confirm that the dual effects of attenuated fatty acid release andincreased fatty acid uptake occur at a single site, the inventorsanalysed two compounds showing either strong or weak inhibitory effectson radiolabel release (isopropyl-pentanoic acid and 4-methyloctanoicacid, respectively) for effects on ³H-AA uptake (FIG. 9). From theseexperiments, enhanced inhibition of radiolabel release corresponded withan elevated uptake of fatty acid into cells as compared with VPA, and areduced effect on release corresponded with a reduced effect on uptake.This data suggest a single, highly structurally-specific site of actionfor VPA and related compounds in the regulation of fatty acidsignalling.

3. Discussion

VPA is used to treat a number of current medical conditions includingepilepsy, Bipolar disorders, and migraine and its role is likely toexpand widely to include cancer (Blaheta et al., 2006), HIV andAlzheimer's (Qing et al. 2008) treatment (reviewed in Lagace et al.,2005, Terbach & Williams, 2009, Bialer & Yagen, 2007), ischemia (Costaet al. 2006), and fatal blood loss (Alam et al. 2009). Understanding howthese conditions are controlled by VPA has proved highly complex sinceit triggers a variety of cellular changes with unknown primary targetsand these changes have not been related to specific clinical conditions,and few structure-function studies have been carried out (reviewed inLagace et al., 2005, Terbach & Williams, 2009, Bialer & Yagen, 2007,Nalivaeva et al., 2009).

3.1 Effect on Phosphoinositol Signalling

The inventors have shown that VPA causes a dose-dependent reduction inPIP and PIP₂ (FIGS. 1a &b) in the biomedical model, Dictyostelium, andthis provides one of the few effects of VPA found to occur in the acutetime period shown to protect against induced seizures (FIG. 1b ; Honack& Loscher, 1992). The vast majority of research into VPA mechanisms hasbeen complicated by long term treatment leading to changes in geneexpression (likely to be mediated by teratogenic effects (Gurvich etal., 2004, Phiel et al., 2001)) or in time periods enabling regulationof multiple indirect targets. Therefore a rapid attenuation ofphosphoinositide signalling provides a significant insight into theacute function of VPA. In light of increased phosphoinositide levelsduring seizures (Van Rooijen et al., 1986), the results shown hereprovide an exciting breakthrough in our understanding of seizurecontrol.

It is very difficult to identify the target(s) of a drug which modulatesphosphoinositide metabolism in vivo in mammalian systems, due to thelarge number of kinases' and phosphatases, and the promiscuous nature oflipid kinases substrate selection. An advantageous approach, facilitatedin Dictyostelium, is to employ isogenic cell lines containing ablatedkinase genes. Since it has previously been shown that VPA attenuatesPIP3 production (Xu et al., 2007), the inventors analysedphosphoinositide turnover in cell lines lacking all type 1 PI3K activity(Hoeller & Kay, 2007), and in two unrelated lipid kinases null mutants(a receptor-linked phosphatidylinositol 5 kinase (rPKA) (Bakthavatsalamet al., 2006); and a nuclear phosphatidylinositol 4/5 kinase activity(PIPKinA) (Guo et al., 2001; FIG. 2). All three mutant cell lines showedlarge and significant reductions in PIP turnover following VPAtreatment, albeit at reduced levels compared to wild type cells,indicating that these enzymes cannot be the direct target of VPA inphosphoinositide attenuation and that testing of further kinases wouldbe of little benefit.

The inositol depletion theory (Berridge et al., 1989) provides awell-supported theory for action of VPA in bipolar disorder phrophylaxis(Williams et al., 2002, Williams, 2005), potentially through theindirect inhibition of the enzyme responsible for de novo inositolbiosynthesis, myo-inositol-1-phosphate synthase (MIP). A stronginhibitory effect on MIP activity has also been shown for a limitednumber of VPA congeners (Shaltiel et al., 2004; Shaltiel et al., 2007),suggesting inositol regulation may be related to seizure control.However, the newly discovered compounds showing strongly improvedseizure control (e.g. 4-MO and nonanoic acid)—do not acutely depleteinositol in the time frame for seizure control, arguing against a rolefor inositol signalling in this clinical treatment. Einat et al., 2008also showed that pharmacological inhibition of MIP (through compoundsstructurally unrelated to VPA) does not control inositol sensitivepilocarpine-induced seizures model. Instead, the data shown here addsweight to the identification of phosphoinositide signalling as playing akey role of seizure occurrence (Backman et al., 2001) and suggests thatVPA's effect on phosphoinositide turnover and seizure control isunrelated to that of inositol depletion.

The majority of research concerning VPA targets and clinical efficacyhas centred around compounds with either a five carbon backbone (with abranch point on the second carbon) or cyclic derivatives (Bialer &Yagen, 2007). Thus the discovery of a novel family of compounds withlonger carbon backbones showing inhibition of phosphoinositide turnoverand seizure control provides a major advance in the development of newtherapeutics. This novel chemical family includes compounds branched atthe second and fourth carbon, suggesting efficacy in compounds withvariable branching, and thus provide a new large family of compounds ofpotential clinical interest.

Here, the inventors tested the efficacy of five compounds within thisnovel family of fatty acid in an in vitro model of epileptiform activitythat is a key model for screening potential antiepileptic drugs (Pireddaet al., 1985). All compounds give rise to a greatly increased butreversible reduction in epileptiform activity in a PTZ model (see FIG.3). This effect is not model specific, since one compound(4-methyloctanoic acid) shows efficacy in a low Mg²⁺ model. However,efficacy in in vitro models does not necessarily imply efficacy in vivo,because other factors such as drug metabolism, brain penetration andaccess to the drug target play an important role. The inventorstherefore tested 4-methyloctanoic acid further in an in vivo model ofstatus epilepticus (FIG. 4), where it proved more potent than VPA. Thismodel has been previously shown to be resistant to phenyloin and torespond only to high doses of other anticonvulsants (Chang et al.,2009). These results therefore suggest that this family of compounds mayprovide a novel seizure control agent with increased efficacy, and sincethey are predicted to not show teratogenicity, they may also providereduced side effects compared to VPA treatment.

These findings identify a mechanism of action of VPA in attenuatingphosphoinositide turnover in the simple biomedical model, Dictyostelium,and have shown that this effect occurs independently of a range ofphosphoinositide kinase enzymes, inositol recycling and depletion. Theinventors used this system to identify a novel family of compoundsshowing increased phosphoinositide attenuation. They then translatedthis simple model-based research to several clinical models of seizurecontrol and show a large increase in efficacy for five of thesecompounds over VPA in a hippocampal slice model of epileptiform activityand for one of these compounds in a whole-animal model of statusepilepticus. These studies thus suggest seizure control efficacy for anovel family of compounds with longer backbone length and variable sidechain length and position compared to VPA. Continued analysis of thebiosynthetic pathways controlling phosphoinositide signalling may giverise to significant advances in understanding epilepsy and otherVPA-treatable disorders such as bipolar disorder and migraine.

3.2 Effect on Fatty Acid Turnover

Previous studies have suggested a possible mechanism of action of VPA isthe attenuation of arachidonic acid turnover (Chang et al., 2001). Herethe inventors have demonstrated in the model Dictyostelium discoideumthat VPA significantly reduces the release of radiolabel following ³H-AAincorporation. Surprisingly, the inventors also observed that the uptakeof ³H-AA was enhanced in the presence of VPA. Since arachidonic acid isnot an endogenous fatty acid in Dictyostelium the inventors alsoverified these results with palmitic acid, a fatty acid found inDictyostelium (Weeks, 1976). The similar results observed for bothpoly-unsaturated and saturated fatty acids suggest a common mechanismfor the modulation of fatty acid metabolism by VPA. The similarity ofthese results shown here and those seen in in vivo animal studies, suchas decreased AA turnover (Chang et al., 2001) and increased lipidaccumulation (Kesterson et al., 1984) suggest that Dictyostelium may bea useful model in the study of VPA induced fatty acid dynamics.

Few studies of fatty acid regulation by VPA in animal models haveexamined a role for VPA in simply elevating fatty acid transport intocells. Thus, to examine this, and since uptake of extra-cellularnutrients in Dictyostelium is regulated by vesicle dynamics(macropinocytosis), the inventors showed that blocking vesicle functionby inhibition action polymerisation did not inhibit the VPA-catalysedincrease in fatty acid uptake. These results are supported by theirprevious results, since they have shown that VPA treatment also inhibitsvesicle dynamics in Dictyostelium (Xu et al., 2007), and these resultstherefore point towards an effect of VPA in regulating fatty acidsignalling which is independent of uptake.

Polyunsaturated fatty acid turnover is primarily regulated byPLA₂-catalysed cleavage from lipids to release free fatty acids, and VPAhas previously been suggested to regulate PLA₂-dependent signalling(Rapoport and Bosetti, 2002). A role for PLA₂ in VPA-dependentsignalling in our model was suggested when a screen for mutantsresistant to the effect of VPA during development revealed that ablationof a single PLA₂ gene gave partial resistance to VPA. This provided anexciting link to a potential clinical function of VPA, since elevatedlevels of PLA₂ have been shown in bipolar disorder patients (Ross etal., 2006) and during seizures (Siesjo et al., 1982; Rintala et al.,1999a; Bazan et al., 2002; Basselin et al., 2003a). However, ablation ofthe single isoform of PLA₂ identified in the genetic screen did notaffect net change in VPA-induced radiolabel release (FIG. 8B). This maybe due to the presence of 18 other PLA₂-like genes present in the genome(Fey et al., 2009), since the activities of these gene products arelikely to hide small changes caused by single gene ablation inwhole-cell assays. However, the identified gene may play a critical,targeted role in Dictyostelium chemotaxis and development—as has beenshown (Chen et al., 2007, Kortholt and van Haastert, 2008), and thuspartially overcome the VPA-related development effects. ThisVPA-dependent inhibition of development may not be visible in the assaysemployed here, since the assay does not differentiate between cell-typespecific function nor do it explore multiple time points overdevelopment.

To further examine PLA₂ signalling in the observed effect of VPA, theinventors used pharmacological inhibitors of PLA₂ activity, and showedthese reduced radiolabel release from ³H-AA labelled cells—thusconfirming that VPA mimics the effect of PLA₂ inhibition in this model.However, this effect of VPA is not through direct PLA₂ inhibition, sincethe VPA-induced increase in fatty acid uptake was not reproduced by PLA₂inhibitors (FIG. 9). This data, suggests that a PLA₂ inhibition-likeeffect of VPA may provide only one aspect of the drugs effect inregulating fatty acid turnover, and confirms that PLA₂ is not theprimary target of VPA in this effect.

If PLA₂ is not the pharmacological target of VPA, the observed partialphenocopying of PLA₂ inhibitors may point to an upstream disruption inlipid metabolism. For example, a reduced activity of fatty acid acyl CoAsynthases may cause a reduced incorporation of radio-labelled fatty acidinto phospholipids, resulting in a reduced release of the radio-labelledfatty acid (thus resembling a PLA₂-inhibitory like effect). Theinventors show that VPA does not inhibit CoA activation of fatty acidseither directly or indirectly. Furthermore, genetic ablation of both offatty acid CoA synthases (FcsA and FcsB) still showed VPA-dependentregulation of AA uptake, thus confirming that fatty acid activation isnot the target of VPA in this effect, in contrast to that reportedearlier at high VPA concentrations (Bazinet et al., 2006b).

Finally, the inventors have employed a SARs study to investigate VPAinduced fatty acid regulation. This approach provides a highly importantinsight into VPA action, since VPA is used in the therapeutic treatmentof a large range of conditions (Terbach and Williams, 2009), withnumerous cellular effects remaining un-associated with each therapeuticrole, and very few of these effects have an identified primary target(Lagace and Eisch, 2005, Terbach and Williams, 2009). SARs studies canbe used to help differential these therapeutic treatments, mechanismsand targets. For example, range of VPA-related compound showing histonedeacetylase inhibition as a cellular function have been shown to causeteratogenicity as a biomedical action, and the structural definition cannow predict teratogenicity in novel compounds (Phiel et al., 2001). Fromthe SARs study for fatty acid turnover, this effect is not related toteratogenicity since some compounds show strong fatty acid regulationbut no predicted teratogenicity (Radatz et al., 1998). Another cellulareffect of VPA in Dictyostelium and mammalian neurons is the inhibitionof inositol based signalling (Williams et al., 2002, Eickholt et al.,2005, Shimshoni et al., 2007). Although some compounds (e.g.isopropyl-pentanoic acid) are strongly active in both inositolsignalling attenuation and fatty acid regulation, these effects are notshared by all compounds, thus indicating these fatty acid- andinositol-based signalling effects are independent. Thus fatty acidregulation, HDAC inhibition and, inositol depletion provide threeindependent mechanisms of action for VPA. Employing VPA structuresshowing increased or decreased activity in fatty acid regulation maytherefore give rise to increased therapeutic activity or a reduction inunwanted side effects in VPA-treatable conditions.

One clinical corollary of this work is shown in VPA-dependent lipiddroplet formation. This effect has been shown in systems ranging from S.cerevisiae (Sun et al., 2007) to hippocampus and neocortex of the ratbrain (Sobaniec-Lotowska, 2005), clearly indicating that the observedeffects reported here are unlikely to be model specific. Furthermore,lipid droplet formation has also been associated with hepatotoxicity,although the mechanism remains unclear (Fujimura et al., 2009). Theidentification of a structural specificity for this effect in lipidregulation provides a potential mechanism for selection of noveltherapeutics lacking this effect. The structural isolation ofVPA-dependent increased lipid accumulation may also go some way toexplaining the weight gain associated with patients undergoing VPAtreatments (Wirrell, 2003, Masuccio et al., 2010, Verrotti et al.,2010).

In conclusion the inventors have described here a model for the study ofVPA-induced fatty acid regulation. PLA₂ inhibition phenocopies some butnot all of these VPA-dependent effects, but PLA₂ is unlikely to be theprimary target of VPA. The role of this effect is likely to function inboth bipolar disorder treatment and seizure control, since increasedPLA₂ activity is implicated in both conditions (Yegin et al., 2002, Raoet al., 2007). Identifying novel structures for this effect, comprisingcarboxylic acids, branched on the C2 position, with short (five carbon)to medium length (nine carbon) backbone and side chain (ethyl or propyl)provides potential new therapies for both conditions. The futuredefinition of the primary site of action for this effect willsignificantly aid our understanding of VPA and related therapeutics.

All documents cited herein are hereby incorporated by reference in theirentirety within this disclosure.

REFERENCES

Ackermann E J, Conde-Frieboes K, Dennis E A, Journal of BiologicalChemistry 270, 445-450 (1995).

Alam et al. Surgery 146, 325-333 (2009)

Armand, V., Louvel, J., Pumain, R., & Heinemann, U. Epilepsy Res. 32,345-355 (1998).

Backman, S. A. et al. Nat. Genet. 29, 396-403 (2001).

Bakthavatsalam, D., Meijer, H. J., Noegel, A. A., & Govers, F. TrendsMicrobiol. 14, 78-382 (2006).

Blaheta, Michaelis, Driever & Cinatl Med. Res. Rev. 25, 383-397 (2005).3. Deubzer et al. Leuk. Res. 30, 1167-1175 (2006)

Balsinde J, Dennis E A, Journal of Biological Chemistry 271, 6758-6765(1996).

Basselin M, Chang L, Bell J M, Rapoport S I, Neuropsychopharmacology 31,1659-1674 (2005).

Basselin M, Chang L, Seemann R, Bell J M, Rapoport S I, J. Neurochem.85, 1553-1562 (2003).

Bazan N G, Tu B, Rodriguez de Turco E B, Prog. Brain. Res. 135, 175-185(2002).

Bazinet R P, Rao J S, Chang L, Rapoport S I, Lee H J, Biol. Psychiatry59, 401-407 (2006a).

Bazinet R P, Weis M T, Rapoport S I, Rosenberger T A, Psychopharmacology(Berl) 184, 122-129 (2006b).

Berridge, M. J., Downes, C. P., & Hanley, M. R. Cell 59, 411-419 (1989).

Bialer, M. & White, H. S, Nat Rev Drug Discov. 9, 68-82 (2010).

Bialer, M. & Yagen, B. Neurotherapeutics. 4, 130-137 (2007).

Boeckeler K, Adley K, Xu X, Jenkins A, Jin T, Williams R S, Eur. J. CellBiol. 85, 1047-1057 (2006).

Chang, P., Chandler, K. E., Williams, R. S., & Walker, M. C. Epilepsia(2009).

Chang M C, Contreras M A, Rosenberger T A, Rintala J J, Bell J M,Rapoport S I, J. Neurochem. 77, 796-803 (2001).

Chapman, A. G., Meldrum, B. S., & Mendes, E. Life Sci. 32, 2023-2031(1983).

Chen C T, Green J T, Orr S K, Bazinet R P Prostaglandins Leukot Essent.Fatty Acids 79, 85-91 (2008).

Chen L, Iijima M, Tang M, Landree M A, Huang Y E, Xiong Y, Iglesias P A,Devreotes P N, Dev. Cell 12, 603-614 (2007).

Chiu C C, Huang S Y, Su K P, Lu M L, Huang M C, Chen C C, Shen W W, Eur.Neuropsychopharmacol 13, 99-103 (2003).

Costa et al. Stroke 37, 1319-1326 (2006)

de Oliveira C A, Mantovani B, Life Science 43, 1825-1830 (1988).

Deutsch, J., Rapoport, S. I., & Rosenberger, T. A. Neurochem. Res. 28,861-866 (2003).

Drancourt M, Bollet C, Carta A, Rousselier P, Int. J. Syst. Evol.Microbiol. 51, 925-932 (2001).

Drayer, A. L., Van Der, K. J., Mayr, G. W., & Van Haastert, P. J. EMBOJ. 13, 1601-1609 (1994).

DSMV IV American Psychiatric Association: Diagnostic and statisticalmanual of mental disorders (American Psychiatric Association, WashingtonD.C., 2000).

Eickholt B J, Towers G J, Ryves W J, Eikel D, Adley K, Ylinen L M,Chadborn N H,

Harwood A J, Nau H, Williams R S, Mol. Pharmacol. 67, 1426-1433 (2005).

Eikel D, Lampen A, Nau H, Chem. Res. Toxicol. 19, 272-278 (2006).

Einat, H., Tian, F., Belmaker, R. H., & Frost, J. W. J. Neural Transm.115, 55-58 (2008).

Eyal S, Yagen B, Shimshoni J, Bialer M, Biochem. Pharmacol. 69,1501-1508 (2005).

Faix J, Kreppel L, Shaulsky G, Schleicher M, Kimmel A R, Nucleic AcidsResearch 32, e143 (2004).

Fey P, Gaudet P, Curk T, Zupan B, Just E M, Basu S, Merchant S N,Bushmanova Y A, Shaulsky G, Kibbe W A, Chisholm R L, Nucleic AcidsResearch 37, D515-519 (2009).

Fujimura H, Murakami N, Kurabe M, Toriumi W, J. Appl. Toxicol. 29,356-363 (2009). Guo, Q. et al. Nat. Med. 5, 101-106 (1999).

Gurvich, N., Tsygankova, O. M., Meinkoth, J. L., & Klein, P. S. CancerRes. 64, 1079-1086 (2004).

Hoeller, O. & Kay, R. R. Curr. Biol. 17, 813-817 (2007).

Honack, D. & Loscher, W. Epilepsy Res. 13, 215-221 (1992).

Holtkamp, M., Tong, X., & Walker, M. C. Ann. Neurol. 49, 260-263 (2001).

Isoherranen, N., Yagen, B., & Bialer, M. Curr. Opin. Neurol. 16, 203-211(2003).

Johnson C. B., Wong E., & Birch E. J. Lipids 12: 340-347 (1977).

Kaufmann, D., Bialer, M., Shimshoni, J. A., Devor, M., & Yagen, B. J.Med. Chem. 52, 7236-7248 (2009).

Keane, P. E., Simiand, J., Mendes, E., Santucci, V., & Morre, M.Neuropharmacology 22, 875-879 (1983).

Kesterson J W, Granneman G R, Machinist J M, Hepatology 4, 1143-1152(1984).

Kim H W, Rapoport S I, Rao J S, Mol. Psychiatry. (2009).

King, J. S. et al. Dis. Model. Mech. 2, 306-312 (2009).

Kortholt A, van Haastert P J, Cell Signal 20, 1415-1422 (2008).

Kuspa A, Loomis W F, Methods Mol. Biol. 346, 15-30 (2006).

Lagace D C, Eisch A J, Psychiatr. Clin. North Am. 28, 399-414 (2005).

Lands W, Crawford C, New York: John Wiley & Sons (1976).

Lio Y C, Reynolds L J, Balsinde J, Dennis E A, Biochim. Biophys. Acta.1302, 55-60 (1996).

Liu, M. J. & Pollack, G. M. P Epilepsia 35, 234-243 (1994).

Loscher, W., Fisher, J. E., Nau, H., & Honack, D. J. Pharmacol. Exp.Ther. 250, 1067-1078 (1989).

Loscher W, Nau H, Neuropharmacology 24, 427-435 (1985).

Maslanski, J. A. & Busa, W. B. Methods in Inositide Research (ed. Irvin,R. F.) 113-126 (Raven Press Ltd., New York, 1990).

Masuccio F, Verrotti A, Chiavaroli V, de Giorgis T, Giannini C,Chiarelli F, Mohn A, J. Child Neural. (2010).

Meunier H, Carraz G, Neunier Y, Eymard P, Aimard M, Therapie 18, 435-438(1963).

Mitchell S M, Poyser N L, Wilson N H, Br. J. Pharmacol. 58, 295P (1976).

Mora, A., Gonzalez-Polo, R. A., Fuentes, J. M., Soler, G., & Centeno, F.Eur I Biochem. 266, 886-891 (1999).

Mora, A., Sabio, G., Alonso, J. C., Soler, G., & Centeno, F. BipolarDisord. 4, 195-200 (2002).

Nalivaeva, N, N., Belyaev, N. D., & Turner, A. J. Trends Pharmacol. Sci.30, 509-514 (2009).

Pawolleck, N. & Williams, R. S. Methods Mol. Biol. 571, 283-290 (2009).

Phiel, C. J. et al. J. Biol. Chem. 276, 36734-36741 (2001).

Piredda, S., Yonekawa, W., Whittingham, T. S., & Kupferberg, H. J.Epilepsia 26, 167-174 (1985).

Qing et al. J. Exp. Med. 205, 2781-2789 (2008)

Radatz M, Ehlers K, Yagen B, Bialer M, Nau H, Epilepsy Research 30,41-48 (1998).

Rao J S, Ertley R N, Rapoport S I, Bazinet R P, Lee H J, J. Neurochem.102, 1918-1927 (2007).

Rao J S, Lee H J, Rapoport S I, Bazinet R P, Mol. Psychiatry. 13,585-596 (2008).

Rapoport S I, J. Nutr. 138, 2515-2520 (2008a).

Rapoport S I, Prostaglandins Leukot Essent. Fatty Acids 79, 153-156(2008b),

Rapoport S I, Bosetti F, Arch. Gen. Psychiatry 59, 592-596 (2002).

Rintala J, Seemann R, Chandrasekaran K, Rosenberger T A, Chang L,Contreras M A, Rapoport S I, Chang M C, Neuroreport 10, 3887-3890(1999).

Ross B M, Hughes B, Kish S J, Warsh J J, Bipolar Disord. 8, 265-270(2006).

Shaltiel, G., Mark, S., Kofman, O., Belmaker, R. H., & Agam, G.Pharmacol. Rep. 59, 402-407 (2007a).

Shaltiel, G., Dalton, E. C., Belmaker, R. H., Harwood, A. J., & Agam, G.Bipolar. Disord. 9, 281-289 (2007b).

Shaltiel, G. et al. Valproate decreases inositol biosynthesis. Biol.Psychiatry 56, 868-874 (2004).

Shimshoni, J. A. et al. Mol. Pharmacol. 71, 884-892 (2007).

Siesjo B K, Ingvar M, Westerberg E, J. Neurochem. 39, 796-802 (1982).

Silva M F, Aires C C, Luis P B, Ruiter J P, Ijlst L, Duran M, Wanders RJ, Tavares de Almeida I, J. Inherit. Metab. Dis. (2008).

Sobaniec-Lotowska M E, Int. J. Exp. Pathol. 86, 91-96 (2005).

Storey, N. M., O'Bryan, J. P., & Armstrong, D. L. Curr. Biol. 12, 27-33(2002).

Sun Q, Bi L, Su X, Tsurugi K, Mitsui K, FEBS Lett. 581, 3991-3995(2007).

Terbach N, Williams R S, Biochem. Soc. Trans. 37, 1126-1132 (2009).

Tokuoka, S. M., Saiardi, A., & Nurrish, S. J. Mol. Biol. Cell 19,2241-2250 (2008).

Vaden, D. L., Ding, D., Peterson, B., & Greenberg, M. L. J. Biol. Chem.276, 15466-15471 (2001).

van Haastert P J, Keizer-Gunnink I, Kortholt A, J. Cell Biol. 177,809-816 (2007).

Van Rooijen, L. A., Vadnal, R., Dobard, P., & Bazan, N. G. Biochem.Biophys. Res. Commun. 136, 827-834 (1986).

Verrotti A, Manco R, Agostinelli S, Coppola G, Chiarelli F, Epilepsia51, 268-273 (2010).

von Lohneysen K, Pawolleck N, Ruhling H, Maniak M, Eur. J. Cell Biol.82, 505-514 (2003).

Walker, M. C. et al. Epilepsia 40, 359-364 (1999).

Weeks G, Biochim. Biophys. Acta. 450, 21-32 (1976).

Williams, R. S. B. Clinical Neuroscience Research 4, 233-242 (2005).

Williams R S, Cheng L, Mudge A W, Harwood A J, Nature 417, 292-295(2002).

Williams R S, Eames M, Ryves W J, Viggars J, Harwood A J, EMBO J. 18,2734-2745 (1999).

Wilson D B, Prescott S M, Majerus P W (1982) Discovery of anarachidonoyl coenzyme A synthetase in human platelets. J Biol Chem 257:3510-3515

Wirrell E C, Pediatr, Neurol. 28, 126-129 (2003).

Worsfold O, Toma C, Nishiya T, Biosens. Bioelectron. 19, 1505-1511(2004).

Xu X, Muller-Taubenberger A, Adley K E, Pawolleck N, Lee V W, WiedemannC, Sihra T S, Maniak M, Jin T, Williams R S, Eukaryot. Cell 6, 899-906(2007).

Yedgar S, Cohen Y, Shoseyov D, Biochim. Biophys. Acta. 1761, 1373-1382(2006).

Yegin A, Akbas S H, Ozben T, Korgun D K, Acta. Neurol. Scand. 106,258-262 (2002).

The invention claimed is:
 1. A method of treating epilepsy, the methodcomprising administering to a subject having epilepsy a therapeuticallyeffective amount of decanoic acid wherein the therapeutically effectiveamount of decanoic acid is about 1 to 1500 μg/kg per dose.
 2. The methodof claim 1, wherein the therapeutically effective amount of the decanoicacid is administered to the subject with one or more of apharmaceutically acceptable carrier, a pharmaceutically acceptableadjuvant or a pharmaceutically acceptable vehicle.
 3. The method ofclaim 1, wherein the therapeutically effective amount of the decanoicacid is administered to the subject orally.